Hospital- and community- acquired infections caused by Staphylococcus aureus are a significant threat to human morbidity and mortality. The emergence of S. aureus strains resistant to all known antimicrobials contributes to this threat by eliminating available treatment options. Identifying novel therapeutic targets against S. aureus is critical to our continued ability to successfully treat these infections. Promising antimicrobial targets are S. aureus systems involved in virulence gene regulation and heme transport as both of these processes are required for successful staphylococcal infection. In this application we describe a novel staphylococcal virulence regulator that recognizes host heme, resulting in a coordinated change in staphylococcal protein expression and heme transport. We have identified two integral components of the S. aureus machinery responsible for this adaptive response: the heme sensor system (Hss) and the heme regulated transporter (Hrt). Preliminary experiments suggest that Hss-mediated activation of Hrt is responsible for protecting S. aureus against the cytoplasmic accumulation of toxic levels of host heme. Animal infection experiments have demonstrated a critical role for Hrt and Hss in staphylococcal pathogenesis, underscoring the importance of heme recognition and transport to staphylococcal virulence. Based on these recent discoveries, new studies are proposed to understand the mechanism and function of Hrt/Hss in host molecule recognition, virulence gene regulation, and small molecule transport during staphylococcal pathogenesis. Preliminary experiments suggest that Hss is a molecular sensor that recognizes heme as a systemic marker of infection. In turn, Hss-mediated activation of HrtAB facilitates small molecule efflux from the cytoplasm protecting S. aureus from toxic metabolite accumulation. This proposal focuses on testing this model. We will utilize genetics, biochemistry and animal infection experiments to (i) determine the role of HssRS in staphylococcal gene regulation, (ii) define the functional consequence of heme-dependent HrtAB activation, and (iii) delineate the role of the Hrt and Hss systems in S. aureus pathogenesis. PUBLIC HEALTH RELEVANCE: Results from these studies will yield mechanistic insight into two newly identified systems involved in an adaptive bacterial response to a host marker of invasive infection. The conservation of these systems in the organisms that cause listeriosis, anthrax, and enterococcal infections enable our results to be extrapolated to multiple human pathogens.